Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA

Abstract

NBU1 is a mobilizable transposon found in Bacteroides spp. Mobilizable transposons require gene products from coresident conjugative transposons for excision and transfer to recipient cells. The integration of NBU1 requires IntN1, which has been identified as a tyrosine recombinase, as well as Bacteroides host factor BHFa. Excision of NBU1 is a more complicated process, involving five element-encoded proteins (IntN1, Orf2, Orf2x, Orf3, and PrmN1) as well as a Bacteroides host factor and a cis-acting DNA sequence. Little has been known about what role the proteins play in excision, although IntN1 and Orf2x have been shown to be the only proteins absolutely required for detectable excision. To determine where IntN1 and Orf2x bind during the excision of NBU1, both proteins were partially purified and tested in DNase I footprinting experiments with the excisive attachment sites attL and attR. The results demonstrate that IntN1 binds to four core-type sites that flank the region of cleavage and strand exchange, as well as six arm-type sites. A unique feature of the system is the location of DR2a and DR2b arm-type sites immediately downstream of the attL core. The DR1a, DR1b, DR3a, and DR3b arm-type sites were shown to be required for in vitro integration of NBU1. In addition, we have identified one Orf2x binding site (O1) on attL as well as a dA+dT-rich upstream element that is required for Orf2x interactions with O1.